Super resolution imaging

ABSTRACT

A detection apparatus that includes (a) an array of responsive pads on a substrate surface; (b) an array of pixels, wherein each pixel in the array has a detection zone on the surface that includes a subset of at least two of the pads; and (c) an activation circuit to apply a force at a first and second pad in the subset, wherein the activation circuit is configured to apply a different force at the first pad compared to the second pad, and wherein the activation circuit has a switch to selectively alter the force at the first pad and the second pad.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/213,340, filed on Mar. 14, 2014, which is a continuation-in-part ofU.S. application Ser. No. 13/835,492, filed Mar. 15, 2013, now U.S. Pat.No. 9,193,998, which is based on, and claims the benefit of, U.S.Provisional Application No. 61/788,954, filed Mar. 15, 2013, nowexpired, each of which is incorporated herein by reference in itsentirety.

BACKGROUND

Embodiments of the present disclosure relate generally to biological orchemical analysis and more particularly to systems and methods usingdetection devices for biological or chemical analysis.

Various protocols in biological or chemical research involve performinga large number of controlled reactions on local support surfaces orwithin predefined reaction chambers. The desired reactions may then beobserved or detected and subsequent analysis may help identify or revealproperties of chemicals involved in the reaction. For example, in somemultiplex assays, unknown analytes having identifiable labels (e.g.,fluorescent labels) may be exposed to thousands of known probes undercontrolled conditions. Each known probe may be deposited into acorresponding location on a surface. Observing any chemical reactionsthat occur between the known probes and the unknown analyte on thesurface may help identify or reveal properties of the analyte. Otherexamples of such protocols include known DNA sequencing processes, suchas sequencing-by-synthesis (SBS) or cyclic-array sequencing.

In some conventional fluorescent-detection protocols, an optical systemis used to direct an excitation light onto fluorescently-labeledanalytes and to also detect the fluorescent signals that may emit fromthe analytes. The resolution of standard imaging techniques isconstrained by the number of pixels available in the detection device,among other things. As such, these optical systems can be relativelyexpensive and require a relatively large bench-top footprint whendetecting surfaces having large collections of analytes. For example,nucleic acid arrays used in genotyping, expression, or sequencinganalyses can require detection of millions of different sites on thearray per square centimeter. Limits in resolution increase cost anddecrease accuracy of these analyses

Thus, there exists a need for higher resolution apparatus and methods,for example, to detect nucleic acid arrays. The present disclosureaddresses this need and provides other advantages as well.

BRIEF SUMMARY

The present disclosure provides a detection apparatus that includes (a)an array of responsive pads on a substrate surface; (b) an array ofpixels, wherein each pixel in the array has a detection zone on thesurface that includes a subset of at least two of the responsive pads;and (c) an activation module to alter a characteristic of a first pad inthe subset and of a second pad in the subset, wherein the activationmodule is configured to apply a different characteristic at the firstpad compared to the second pad, and wherein the activation module has aswitch to selectively alter the characteristic at the first pad comparedto the second pad.

In particular embodiments, a detection apparatus can include (a) anarray of electrically responsive pads on a substrate surface; (b) anarray of pixels, wherein each pixel in the array has a detection zone onthe surface that includes a subset of at least two of the electricallyresponsive pads; and (c) an activation circuit to apply an electricfield at a first pad in the subset and a second pad in the subset,wherein the activation circuit is configured to apply a differentelectric field at the first pad compared to the second pad, and whereinthe activation circuit has a switch to selectively alter the electricfield at the first pad compared to the second pad.

The disclosure also provides a nucleic acid sequencing system. Thesystem can include (a) a detection apparatus having (i) an array ofresponsive pads on a substrate surface; (ii) an array of pixels, whereineach pixel in the array has a detection zone on the surface thatincludes a subset of pads; and (iii) an activation module to alter acharacteristic of the pads in the subset individually, wherein theactivation module is configured to apply a different characteristic to afirst pad of the subset compared to the other pads of the subset; (b) areadout circuit to acquire signals from the array of pixels; (c) acontrol module that directs the readout circuit to acquire signals fromeach of the pixels during a sensing period and that directs theactivation module to sequentially apply different characteristic at thepads during the sensing period; and (c) a processing module thatcorrelates (i) the signals acquired from the pixels during the sensingperiod and (ii) the sequential application of the differentcharacteristics at the pads during the sensing period, in order todistinguish a sequence of signals for each of the pads.

In particular embodiments, a nucleic acid sequencing system can include(a) a detection apparatus having (i) an array of electrically responsivepads on a substrate surface; (ii) an array of pixels, wherein each pixelin the array has a detection zone on the surface that includes a subsetof four of the pads; and (iii) an activation circuit to apply anelectric field to the pads in the subset individually, wherein theactivation circuit is configured to apply a different electric field ata first pad of the subset compared to the other pads of the subset; (b)a readout circuit to acquire signals from the array of pixels; (c) acontrol module that directs the readout circuit to acquire signals fromeach of the pixels during a sensing period and that directs theactivation circuit to sequentially apply different electric fields atthe four pads during the sensing period; and (c) a processing modulethat correlates (i) the signals acquired from the pixels during thesensing period and (ii) the sequential application of the differentelectric fields at the four pads during the sensing period, in order todistinguish a sequence of signals for each of the pads.

The disclosure further provides a method of detecting analytes. Themethod can include the steps of (a) providing a detection apparatushaving an array of responsive pads and an array of pixels, wherein eachpixel in the array has a detection zone that includes a subset of atleast two of the responsive pads, wherein the two pads include differenttarget analytes, respectively; (b) acquiring signals from each of thepixels while selectively applying a unique characteristic at a first ofthe two pads to preferentially produce signal from a first of thedifferent target analytes compared to a second of the target analytes,thereby preferentially acquiring signals from the first of the targetanalytes compared to the second of the target analytes; and (c)acquiring signals from each of the pixels while selectively applying theunique characteristic at the second of the two pads to preferentiallyproduce signal from the second of the different target analytes comparedto the first of the target analytes, thereby preferentially acquiringsignals from the second of the target analytes compared to the first ofthe target analytes.

In particular embodiments, a method of detecting analytes can includethe steps of (a) providing a detection apparatus having an array ofelectrically responsive pads and an array of pixels, wherein each pixelin the array has a detection zone that includes a subset of at least twoof the electrically responsive pads, wherein the two pads includedifferent target analytes, respectively; (b) acquiring signals from eachof the pixels while selectively applying an electric field at a first ofthe two pads to preferentially produce signal from a first of thedifferent target analytes compared to a second of the target analytes,thereby preferentially acquiring signals from the first of the targetanalytes compared to the second of the target analytes; and (c)acquiring signals from each of the pixels while selectively applying anelectric field at the second of the two pads to preferentially producesignal from the second of the different target analytes compared to thefirst of the target analytes, thereby preferentially acquiring signalsfrom the second of the target analytes compared to the first of thetarget analytes.

The present disclosure also provides a detection apparatus that includes(a) an array of responsive pads on a substrate surface, wherein eachresponsive pad includes a nucleic acid feature of a plurality of nucleicacid features in the array, wherein a first subset of nucleic acidfeatures in the plurality of nucleic acid features have a firstuniversal sequence and different target sequences, wherein a secondsubset of nucleic acid features in the plurality of nucleic acidfeatures have a second universal sequence and different targetsequences, wherein the first universal sequence is different from thesecond universal sequence; (b) an array of pixels, wherein each pixel inthe array has a detection zone on the surface that includes at least twonucleic acid features of the plurality of nucleic acid features, the atleast two nucleic acid features including a nucleic acid from the firstsubset of nucleic acid features and a nucleic acid from the secondsubset of nucleic acid features; and (c) an activation module to alter acharacteristic of a pad in the first subset and of a pad in the secondsubset, wherein the activation module is configured to apply a differentcharacteristic at the pad in the first subset compared to the pad in thesecond subset, and wherein the activation module has a switch toselectively alter the characteristic at the pads in the first and secondsubsets. Optionally, the detection apparatus can be included in anucleic acid sequencing system that also includes (I) a readout circuitto acquire signals from the array of pixels; (II) a control module thatdirects the readout circuit to acquire signals from each of the pixelsduring a sensing period and that optionally directs the activationcircuit to sequentially actuate different responsive pads in each of thedetection zones during the sensing period; and (III) a processing modulethat optionally correlates (i) the signals acquired from the pixelsduring the sensing period and (ii) the sequential actuation of thedifferent responsive pads during the sensing period, in order todistinguish a sequence of signals for each of the pads.

Also provided is a method of detecting target nucleic acids, includingthe steps of (a) providing a substrate comprising an array of pads, thearray of pads including a first subset of the pads and a second subsetof the pads; (b) delivering a first solution to the substrate, whereinthe first solution includes a first plurality of different targetnucleic acids that selectively attach to the first subset of padscompared to the second subset of pads; (c) delivering a second solutionto the substrate, wherein the second solution includes a secondplurality of different target nucleic acids that selectively attach tothe second subset of pads compared to the first subset of pads; and (d)detecting the substrate using an apparatus having an array of pixels,wherein each pixel in the array has a detection zone that includes (i)at least one of the target nucleic acids that is attached to a pad ofthe first subset of pads, and (ii) at least one of the target nucleicacids that is attached to a pad of the second subset of pads.

In some embodiments, a method of detecting nucleic acids can include thesteps of (a) providing a substrate comprising an array of pads, thearray of pads including a first subset of the pads and a second subsetof the pads; (b) contacting a solution of adapter nucleic acids with thesubstrate while selectively actuating one or both of the subsets ofresponsive pads, wherein the adapter nucleic acids attach to responsivepads of the one or both subsets that are selectively actuated; (c)contacting a first solution with the substrate, wherein the firstplurality of different target nucleic acids attach to responsive pads ofthe first subset; (d) contacting a second solution with the substrate,wherein the second plurality of different target nucleic acids attach toresponsive pads of the second subset, wherein the responsive pads of oneor both of the first and second subset are attached to the adapternucleic acids; and (e) detecting the substrate using an apparatus havingan array of pixels, wherein each pixel in the array has a detection zonethat includes (i) at least one of the target nucleic acids that isattached to a pad of the first subset of pads, and (ii) at least one ofthe target nucleic acids that is attached to a pad of the second subsetof pads.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a diagrammatic view of a detection apparatus having anarray of pixels and an array of electrically responsive pads.

FIG. 2 shows a diagrammatic view of a detection apparatus having anarray of pixels and an array of electrically responsive pads, whereinthe pads each have multiple target analyte features.

FIG. 3 shows side views of (A) an array of pixels and an array ofelectrically responsive pads integrated into a substrate, and (B) anarray of pixels in a detection unit positioned to detect electricallyresponsive pads on a substrate that is separate from the detection unit.

FIG. 4 shows a diagram of a detection system.

FIG. 5 shows a diagram of a detection apparatus that differentiallydetects fluorescent analytes from two pads in the detection zone of asingle pixel by selectively attracting a fluorescence quencher to one ofthe pads.

FIG. 6 shows fluorescence intensity modulation when applying multiplecycles of charge on an electrically responsive pad having afluorescently labeled nucleic acid attached to the pad via a gel.

FIG. 7 shows (A) a template nucleic acid hybridized to a primer having afluorophore at the 3′ end and a quencher tethered to the 5′ end via alinker arm; (B) the quencher moiety drawn toward the fluorophore by apositive electric field and (C) the quencher moiety repelled away fromthe fluorophore by a negative electric field.

DETAILED DESCRIPTION

This disclosure provides apparatus and methods for super resolutionimaging. The resolution of standard imaging techniques is constrained bythe number of pixels available in the detection device, among otherthings. Standard imaging techniques provide, at best, a single pixel fordetection of each feature in an object. Often several pixels must beused to collect signal from each feature in order to obtain sufficientsignal to noise. In contrast, the super resolution imaging methodsprovided by the current disclosure break the one-pixel-per-featurebarrier and allow several features to be distinguished by a singlepixel. This leads to advantages of reducing the optics hardware requiredto detect an object of a given size and complexity. This can alsoincrease the resolution of a detection apparatus beyond the resolutionof the optics module being used. Thus, costs incurred in manufacturingand using optics hardware can be reduced substantially.

In embodiments set forth herein, two features can be resolved with asingle pixel by differential treatment of the features to render one ofthe features detectable in a first state and the other featuredetectable in another state. By extension, several features can beresolved by a single pixel by sequentially actuating individual featuresto a detectable state while the other features are in an undetectablestate.

A particularly useful application of the present apparatus and methodsis the detection of analytes, such as nucleic acids, on solid supports,such as arrays of features to which the analytes are attached. Inparticular embodiments, the solid support has several responsive padsthat are in the detection zone of a particular pixel and each pad has adifferent target analyte attached. The pads can be switchable betweendifferent states such that a single pad is in a first state thatproduces signal from the respective analyte; meanwhile the other padsare in a different state such that the analytes at these other pads donot produce a detectable signal. For example, the single pad may have anelectric field that attracts a detectable label (e.g. a fluorophore),removes a detection inhibitor (e.g. a fluorescence quencher) or induceselectrochemical luminescence. By sequentially switching the states (e.g.strength, polarity or presence of an electric field) at the pads thatare in the detection zone, the different analytes can be individuallyinduced to produce signal. By detecting the pads in the different statesthe pixel can achieve super resolution detection of several differentanalytes in its detection zone. An accounting of which pads wereactuated at different times during acquisition of signal by the pixelwill allow the respective analytes to be distinguished.

Alternatively or additionally, to differential treatment of two featuresduring detection events, two features can be distinguished by one pixelbased on physical or chemical differences that were imparted to thefeatures prior to the detection events. Thus, the two features need notbe switched between different states during or between detection steps.For example, each of the two different target nucleic acid featureswithin the detection zone of the same pixel can have different primingsequences such that they can be distinguished by hybridizing differentprimers to each feature and/or extending different primers at eachfeature. The hybridization and/or extension can occur at the respectivefeatures at different times. In multiplex formats, the priming sites canbe universal sequences such that a first subset of features in the arrayshare a first universal priming site sequence and a second subset offeatures in the array share a second universal priming site sequence. Inthis multiplex format, individual pixels can have a detection zone thatincludes a feature from each subset. Thus each pixel can be capable ofsuper resolution detection based on differential primer hybridization.For example, different primers can be delivered sequentially forseparate detection of features at different times.

By way of further illustrative example, two pads that are in thedetection zone of the same pixel can differentially capture targetnucleic acids. Differential capture can be facilitated by use ofspecific capture probes that are attached at respective pads and thatare selective for different target nucleic acids. Nanofabricationmethods, such as those used in the manufacture of nucleic acidmicroarrays, can be used to place different capture probes at separatefeatures within the detection zone of the same pixel. Alternatively, thesame capture probes can be present at both pads in the detection zone,but the pads can be differentially activated in the presence ofdifferent adapter nucleic acids such that the capture probes on the twodifferent pads hybridize to different adapter nucleic acids. Thedifferent adapter nucleic acids can include a common sequence that iscomplementary to the capture probes and different selective capturesequences that are complementary to different target nucleic acids. Assuch, these adapters can convert a universal surface to one that iscapable of selective target nucleic acid capture.

Similarly, different nucleic acids present on two pads can serve ashybridization sites for different amplification primers or differentdetection primers. Different amplification primers can be used fordifferential amplification of target nucleic acids before or during adetection event. In this example a first amplification primer canamplify a subset of target nucleic acid templates that have a firstuniversal priming site that is complementary to the first amplificationprimer. A second amplification primer can amplify a subset of targetnucleic acid templates that have a second universal priming site that iscomplementary to the second amplification primer. The differentamplification primers can be delivered to an array of pads at differenttimes to create colonies at a first subset of the pads for a firstsensing period followed by creation of colonies at a second subset ofpads for a second detection event. Different extension primers can beused for differential detection of target nucleic acids for example intechniques such as sequencing-by-synthesis, sequencing-by-ligation,single base extension, oligonucleotide ligation, allele specific primerextension or the like. In this example a first extension primer can beused to detect a subset of target nucleic acid templates that have afirst universal priming site that is complementary to the firstextension primer and a second extension primer can detect a subset oftarget nucleic acid templates that have a second universal priming sitethat is complementary to the second extension primer.

As exemplified in further detail below, super resolution imaging can beachieved using electrically responsive pads, for example, to achieveelectric field assisted transport of charged species to or from the pad.Other types of responsive pads can be used similarly in an apparatus ormethod set forth herein. For example, a responsive pad can alter otherforces to create non-diffusive forces on target analytes, probes orother materials of interest (whether charged or not). Non-diffusiveforces can be provided by an external source such as those that producean electrical or magnetic field, or an agent that imposes molecularcrowding or chemical gradients within a reaction volume. For example,magnetic or optical forces can be used to increase the localconcentration of desired materials at a pad in an array of pads or todecrease the local concentration of the materials at a particular pad.In such cases, the materials can include a magnetic tag or optical tagthat can be manipulated by such forces. Other useful responsive pads canalter detection properties of an analyte at the pad, for example, byinducing chemical changes at or near the pad that activate, inhibit,destroy or create a detectable label. Such pads can be used to replaceelectrically responsive pads exemplified in several embodiments of theapparatus and methods set forth herein.

Particular embodiments of the super resolution apparatus and methods setforth herein provide the advantage of reducing the number of differentexcitation lines and/or fluorophore labels required to observe ananalyte. In many fluorescent detection systems, analytes are labeledwith multiple different fluorophore labels, respectively, and the labelsare distinguished by use of multiple different excitation wavelengths.Using the super resolution apparatus and methods of the presentdisclosure, responsive pads (e.g. electrically responsive pads) canremove the necessity for multiple fluorophores and multiple excitationlines. For example, instead of using two different fluorophores and twodifferent lasers to distinguish two analytes in a detection area, tworesponsive pads can occur in the detection area. The pads, althoughhaving different analytes, can be labeled with the same fluorophore andexcited with the same excitation laser. However, it will be understoodthat in some embodiments multiple excitation lines can be used incombination with super resolution to further expand the number ofdifferent analytes detected by a given pixel. More specifically,multiple features in the detection zone of a pixel can be individuallyactuated to produce or inhibit signals, and features that are active canbe excited sequentially with different radiation lines. As such, a pixelcan distinguish a number of analytes that is equivalent to themathematical product of the number of responsive pads in the detectionzone of the pixel multiplied by the number of excitation lines thatproduce emission at each of the pads.

An alternative to actuated pads is to use pads that are manufactured tohave different physical or chemical properties. Taking as an example,nucleic acid-based techniques, two pads that are, or will be, present inthe detection zone of the same pixel can be nanofabricated to includedifferent capture nucleic acids. A first feature can have a firstcapture sequence and a second feature can have a second capturesequence. In array formats, a first subset of features can have captureprobes with a first universal sequence and a second subset of featurescan have capture probes with a second universal sequence.

The present disclosure provides a detection apparatus that includes (a)an array of electrically responsive pads on a substrate surface; (b) anarray of pixels, wherein each pixel in the array has a detection zone onthe surface that includes a subset of at least two of the electricallyresponsive pads; and (c) an activation circuit to apply an electricfield at a first pad in the subset and a second pad in the subset,wherein the activation circuit is configured to apply a differentelectric field at the first pad compared to the second pad, and whereinthe activation circuit has a switch to selectively alter the electricfield at the first pad compared to the second pad.

As used herein, the term “electrically responsive pad” means an area onthe surface of a substrate that produces an electric field. The area onthe surface can form an interface between the substrate and a fluid thatis in contact with the substrate. Thus, the electric field can attractelectrically charged species from the fluid to the pad or the field canrepel electrically charged species from the pad. The direction of activetransport to or from the pad will depend upon the charge of the speciesin solution and the charge of the field at or near the pad.Specifically, positively charged species, such as positively chargedfluorophores or other optical labels, receptors, ligands, quenchers,labeled probes or the like, can be attracted to the pad by inducing anegative charge at or near the pad. Conversely, negatively chargedspecies, such as nucleic acids, nucleotides, or negatively chargedfluorophores, optical labels, receptors, ligands, quenchers, labeledprobes or the like, can be attracted to the pad by inducing a positivecharge at or near the pad.

As used herein, the term “electric field,” when used in reference to anelectrically responsive pad, means the effect produced by the existenceof an electric charge on the pad or in the volume of a medium thatsurrounds the pad. A charge placed in the volume of a medium has a forceexerted on it. Electric fields are created by differences in voltage:the higher the voltage, the stronger will be the resultant field. Incontrast, magnetic fields are created when electric current flows: thegreater the current, the stronger the magnetic field. An electric fieldwill exist whether or not current is flowing. Electric fields can bemeasured in Volts per meter (V/m) or similar units. Electric fieldstrength of about 5 V/cm or higher, up to practical limits of Jouleheating and dielectric breakdown limits, are particularly useful tocause movement of charged particles and species in the present methodsand apparatus. In particular embodiments, the maximum upper value forthe field strength is about 1000 V/cm.

Electrical potential greater than the redox potential of water, roughly1.23 V, will cause electrolysis of water. In some embodiments, such asthose using aqueous fluids, the applied voltages can be in the range of−1 V to +1 V. In some embodiments, it may be beneficial to employ acommon ground counter electrode separating the pads to minimizediffusion of labels, target analytes or other substances from one pad toanother.

Particular embodiments use an AC electric field with DC bias to attractor repel charged species. Exemplary configurations for applying an ACelectric field with DC bias are set forth in U.S. Pat. No. 8,277,628,which is incorporated herein by reference.

As used herein, the term “different electric field,” when used withrespect to a reference electric field, includes, for example, a fieldhaving opposite charge compared to the reference field, no chargecompared to the reference field, greater or lesser charge compared tothe reference field, a DC induced charge compare to an AC induced chargeat the reference field, or an AC induced charge compared to a DC inducedcharge at the reference field.

The conductive surfaces of a pad can be metallic (e.g. gold, titanium,indium tin oxide) or semiconducting in nature. In some embodiments, itmay be desirable to use an electrical conductor that is transparent toradiation used in an optical detection step. Examples of opticallytransparent electrode materials include, but are not limited to metaloxides such as indium tin oxide, antimony doped tin oxide, and cadmiumtin oxide. This is particularly useful in configurations where the padoccurs between a target analyte and a pixel that will detect the analyteand/or when the pad occurs between a target analyte and an excitationsource used in a fluorescence technique.

In particular embodiments, electrically responsive pads can beelectrically coupled to a power source to produce an electric chargethat attracts target nucleic acids or other substances. In oneconfiguration, a positive charge at the pad can attract nucleic acidsvia the negatively charged sugar-phosphate backbone. Exemplary methodsand apparatus for using e-field assist to attract nucleic acids or othersubstances to sites of an array are described in U.S. Pat. No.8,277,628, which is incorporated herein by reference. Alternatively,pads of an array can be electrically coupled to a power source toproduce an electric charge that inhibits binding of or removes targetnucleic acids or other substances from the pads. In one configuration, anegative charge at the pads can repel nucleic acids via the negativelycharged sugar-phosphate backbone.

A low conductivity and low ionic buffers, such as 10-100 mM histidine,can be used to promote electrophoretic transport of a label, targetanalyte or other substance to or from an electrically responsive pad. Alow ionic strength buffer also provides a benefit of increasing theDebye length, effectively increasing the spatial extent of the electricfield in the solution above the activated pad.

Although several methods and apparatus of the present disclosure areexemplified with regard to electrically responsive pads, it will beunderstood that other responsive pads can be used in place of these. Asused herein, the term “responsive pad” means an area on the surface of asubstrate that can be physically or chemically manipulated to alter asurface characteristic. The area on the surface can form an interfacebetween the substrate and a fluid that is in contact with the substrate.A change in a surface characteristic of the pad can induce a change inthe fluid that contacts the pad. Exemplary characteristic that can bealtered include, but are not limited to, electric field, electriccurrent, temperature, magnetic field, or a chemical property such as pH,redox potential, hydrophobicity, hydrophilicity or chemical reactivity.A characteristic of a responsive pad can be altered to change thedirection of transport of a material or substance to or from the pad. Acharacteristic of a responsive pad can also be altered to change thechemical composition or structural integrity of a material or substanceat the pad.

As used herein, the term “electrowetting control pad” refers to a pad orarea comprising an electrode covered by a hydrophobic layer. Thehydrophobic layer becomes hydrophilic upon activation of theelectrowetting control pad. The size of the electrowetting control padis generally approximately equivalent to the size of the electrode. Inparticular embodiments, two or more electrodes can be configured to bein the detection zone of the same pixel. More particularly, featureslocated on two or more different electrowetting control pads can belocated within the detection zone of the same pixel.

As used herein, the term “transport” refers to movement of a moleculethrough a fluid. The term can include passive transport such as movementof molecules along their concentration gradient (e.g. passivediffusion). The term can also include active transport whereby moleculescan move against their concentration gradient or at an increased rate ofpassage along their concentration gradient. Thus, transport can includeapplying energy to move one or more molecule in a desired direction orto a desired location such as a pad in an array of pads. Activetransport can be provided by an external source such as those thatproduce electric or magnetic fields, or an agent that imposes molecularcrowding or chemical gradients within a reaction volume. For example,magnetic or optical forces can be used to increase the localconcentration of amplification reagents. In such cases, one or moreamplification reagents can include a magnetic tag or optical tag thatcan be manipulated by such forces.

An array of pixels can be configured such that each pixel in the arrayhas a detection zone that includes a subset of at least two pads orother features to be detected. As used herein, the term “detectionzone,” when used in reference to a pixel, means a location that issimultaneously observed by the pixel. The location can be, for example,a volume of space or area on a surface. For example, a detection zonecan include an area on the surface of an array of pads that includes asubset of the pads. For example, the detection zone of an individualpixel can include at least 2, 3, 4, 5, 10 or more pads or features. Thenumber of pads or features in a detection zone can be selected to suitthe size of the pixel, the size of the detection zone for the pixel (forexample, as influenced by optics between the pixel and the features orpads observed), the size of the features or pads, or the size of anyanalytes to be detected by the pixel. In particular embodiments, amaximum number of pads or features in a detection area can be 10, 5, 4,3 or 2.

As used herein, the term “each,” when used in reference to a collectionof items, is intended to identify an individual item in the collectionbut does not necessarily refer to every item in the collection.Exceptions can occur if explicit disclosure or context clearly dictatesotherwise. Thus, reference to each pixel in an array having a detectionzone that includes a subset of at least two pads, means that at leastone pixel in the array has a detection zone that includes the subset ofpads. Although all of the pixels in the array may have a similarlyconfigured detection zone, not all of the pixels in the array need to beso configured. Rather some pixels may not have any pads in theirrespective detection zone or some pixels may have only one pad in therespective detection zone.

A diagrammatic representation of a relationship between an array ofpixel detection zones and an array of responsive pads that providessuper resolution imaging is shown in FIG. 1. Detection apparatus 10 hasa 5×5 array of pixel detection zones 1 and a 4×4 array of responsivepads 2. The arrays are offset such that each pixel detection zone 1includes four different pads 2. For example pixel detection zone 1 aincludes pads 2 a, 2 b, 2 c and 2 d. In this exemplary configuration,each pad 2 occurs in the detection zone of four different pixels 1. Forexample, pad 2 a is in the detection zone of pixels 1 a, 1 b, 1 c and 1d. In the exemplified configuration of FIG. 1, each detection zone issquare and each pad occurs in a corner of four of the detection zones.Furthermore each pad is exemplified as being square and each detectionzone includes a corner of four of the pads. As used herein, the term “ina corner” means at or near the intersection of two vertices. Forexample, an object that occurs in all or part of one quadrant of asquare area can be considered to be in the corner of the square area.

In the exemplary configuration of FIG. 1, the array of pads has roughlythe same pitch (center to center spacing for the pads) as the pitch forthe array of pixels. Also the pads have an area that is roughlyequivalent to the area of each pixel's detection zone (although theareas for any given pad and detection zone only partially overlap due tothe offset between the two arrays). Also, the pads are adjacent to eachother and the pixel detection zones are adjacent to each other. Thus,the pads and pixels have the same spacing. This configuration isexemplary as the pitch, areas and spacing can differ for the two arrays.

In some embodiments, the pads can have a pitch that is less than thepitch for the detection zones. This can allow greater than four pads perdetection zone in the rectilinear configuration such as the oneexemplified in FIG. 1. Depending upon the desired use, the array of padson a surface can have a pitch that is no greater than the pitch of thedetection areas, no greater than half the pitch of the detection areas,no greater than a quarter of the pitch of the detection areas or nogreater than a tenth of the pitch of the detection areas, or smallerpitch.

The areas for individual pads can be substantially smaller than the areaof each detection zone. In particular embodiments this can allow eachdetection zone to include several pads while each pad is present in onlya single detection zone (in contrast to the example of FIG. 1 where eachpad is present in four detection zones). The area for individual pads ona surface can be at most the same as the detection area, at most 75% ofthe detection area, at most 50% of the detection area, at most 25% ofthe detection area or at most 10% of the detection area or smaller.

The pads are exemplified as being juxtaposed to each other and pixeldetection zones are also exemplified as being juxtaposed to each otherin FIG. 1. In alternative embodiments, spacing can occur between pads orbetween pixel detection zones. The spacing for one of the arrays can beequivalent or different compared to the other array. For example, thespacing between the pads on a surface can be greater or smaller than thespacing between the pixel detection areas on the surface.

Although detection areas and pads are exemplified above as being square,it will be understood that reactive pads on a surface and/or pixeldetection areas on the surface can have other shapes including, but notlimited to, rectangular, circular, oval, hexagonal, triangular,polygonal or the like. A particularly illustrative example is ahexagonally packed array. Hexagonal packing is a particularly usefulconfiguration for close packing of round pads or round detection areas.In an exemplary hexagonal arrangement, each pixel in an array can have adetection area that is round and includes a subset of seven responsivepads on the surface of a substrate. Furthermore in this packing, eachpad can be included in the detection areas for three of the pixels. Thuseach detection area can be considered as approximating a hexagon andeach pad can be considered to cross into three detection zones at acorner of each hexagon. Alternatively, each pad can have an area that isround and can be included in seven detection areas on the surface of asubstrate. Furthermore in this packing, each pixel in the array can havea detection area on the surface that includes three of the pads. Thus,each pad can be considered as approximating a hexagon and each detectionarea can be considered to cross into three detection zones at a cornerof each hexagon.

An apparatus or system of the present disclosure can include an array ofpixels that are in a complementary metal oxide semiconductor (CMOS)sensor, charge coupled device (CCD) sensor or other digital cameras. Insome embodiments the pixels are used to detect chemiluminescence,electrochemical luminescence or other optical signals that do notrequire excitatory radiation. However, many embodiments utilizefluorescence based detection. In these cases, an apparatus or system ofthis disclosure can include an optical excitation assembly. Radiationcan be provided from a laser, light emitting diode (LED), or otherappropriate radiation source. Furthermore the radiation can beconditioned by optics to reflect, filter, shape, direct or otherwisemanipulate the excitation radiation.

Embodiments described herein may utilize a step-and-shoot procedure inwhich different portions of an array of pads are individually detectedor imaged between (or after) relative movements of the detector andsample. For example, each area of the array can be excited with a laseror other appropriate radiation source and emission can be detected usingan array of pixels configured for super resolution imaging. Examples ofstep-and-shoot optical components that can be modified for superresolution imaging in accordance with the present disclosure are setforth in US Pat. App. Pub. No. 2012/0270305 A1, which is incorporatedherein by reference. Embodiments described herein may utilize a scanningprocedure in which different portions of an array of pads are detectedor imaged during movement between the pads and optical components. Insome embodiments, the imaging assembly includes a scanning time-delayintegration (TDI) system. Furthermore, the imaging sessions may includeline-scanning one or more samples such that a linear focal region oflight is scanned across the array of pads. Some methods of line-scanningthat can be modified for super resolution imaging in accordance with thepresent disclosure are described, for example, in U.S. Pat. No.7,329,860 and U.S. Pat. Pub. No. 2009/0272914, each of which isincorporated herein by reference. Scanning may also include moving apoint focal region of light in a raster pattern across the array ofpads. Whether using step-and-shoot, scanning, static image collection orother configurations, embodiments can be configured for epi-fluorescentimaging or total-internal-reflectance-fluorescence (TIRF) imaging.Exemplary optical components and arrangements that can be modified forsuper resolution imaging in this regard are set forth in U.S. patentapplication Ser. No. 13/766,413; US Pat. App. Pub. Nos. 2010/0111768 A1and 2012/0270305 A1; and U.S. Pat. Nos. 7,329,860 and 8,241,573, each ofwhich is incorporated herein by reference.

Certain embodiments include objective lenses having high numericalaperture (NA) values. Exemplary high NA ranges for which embodiments maybe particularly useful include NA values of at least about 0.6. Forexample, the NA may be at least about 0.65, 0.7, 0.75, 0.8, 0.85, 0.9,0.95, or higher. Those skilled in the art will appreciate that NA, beingdependent upon the index of refraction of the medium in which the lensis working, may be higher including, for example, up to 1.0 for air,1.33 for pure water, or higher for other media such as oils. However,other embodiments may have lower NA values than the examples listedabove. Image data obtained by the optical assembly may have a resolutionthat is between 0.1 and 50 microns or, more particularly, between 0.1and 10 microns. Optical assemblies may have a resolution that issufficient to individually resolve the features or sites that areseparated by a distance of less than 15 μm, 10 μm, 5 μm, 2 μm, 1 μm, 0.5μm, or less.

In general, the NA value of an objective lens is a measure of thebreadth of angles for which the objective lens may receive light. Thehigher the NA value, the more light that may be collected by theobjective lens for a given fixed magnification. This is because thecollection efficiency and the resolution increase. As a result, multiplepads or features may be distinguished more readily when using objectiveslenses with higher NA values. Therefore, in general, a higher NA valuefor the objective lens may be beneficial for imaging.

The size of the pads (or target analyte features) and/or spacing betweenthe pads (or target analyte features) can vary such that arrays can behigh density, medium density or low density. High density arrays arecharacterized as having pads (or features) separated by less than about15 μm. Medium density arrays have pads (or features) separated by about15 to 30 μm, while low density arrays have pads (or features) separatedby greater than 30 μm. An array useful in some embodiments can have pads(or features) that are separated by less than 100 μm, 50 μm, 10 μm, 5μm, 1 μm, or 0.5 μm. An apparatus or method of the present disclosurecan be used to image an array of pads or features at a resolutionsufficient to distinguish pads or features at the above densities ordensity ranges. In particular embodiments the size of the responsivepads can be smaller than the resolution limit of the detector componentthat is used. In this way, the super resolution methods and apparatus ofthe present disclosure can distinguish target analyte features at aresolution beyond the limits of the detector component.

FIG. 2 shows a diagrammatic representation of an array of pixeldetection zones 1, an array of responsive pads 2 and an array of targetanalyte features 3 that are configured to provide super resolutionimaging of the features 3. Each target analyte feature 3 is shown asbeing in the corner of a pad 2 that occurs in a detection zone of apixel 1. For example pixel detection zone 1 a includes a target analytefeature 3 that is present on each of pads 2 a, 2 b, 2 c and 2 d,respectively. In this exemplary configuration (i) each target analytefeature occurs in a single detection zone, (ii) each detection zoneincludes four different target analyte features and each target analytefeature is present on a different responsive pad than the other targetanalyte features in the respective detection zone. Thus, each pixel candistinguish the four target analytes based on differential actuation ofthe four pads, respectively.

In an alternative embodiment to that exemplified in FIG. 2, a targetanalyte can occur in multiple detection zones. For example, one canenvision a situation where the four target analyte features 3 on pad 2 aare replaced with a single feature that occupies the entire surface areaof pad 2 a. As such the target analyte feature will occur in the fourdetection zones 1 a, 1 b, 1 c and 1 d. In this situation activation ofsignal at pad 2 a will result in detection by all four of the pixels.However, in this configuration each pixel will still include four padsin its detection zone.

A detection zone can include multiple target analyte features, each on aseparate responsive pad from others in the detection zone. For example,the detection zone of an individual pixel can include at least 2, 3, 4,5, 10 or more target analyte features, each on separate responsive pads.The number of target analyte features in a detection zone can beselected to suit the size of the pixel, the size of the detection zonefor the pixel (for example, as influenced by optics between the pixeland the features or pads observed), the size of the pads and the size ofthe target analyte features. In particular embodiments, a maximum numberof target analyte features in a detection area can be 10, 5, 4, 3 or 2.

Any of a variety of target analytes that are to be detected,characterized, or identified can be used in an apparatus, system ormethod set forth herein. Exemplary analytes include, but are not limitedto, nucleic acids (e.g. DNA, RNA or analogs thereof), proteins,polysaccharides, antibodies, epitopes, receptors, ligands, enzymes (e.g.kinases, phosphatases or polymerases), small molecule drug candidates,cells, viruses, organisms, or the like. An array of pads can includemultiple different species from a library of analytes. For example, thespecies can be different antibodies from an antibody library, nucleicacids having different sequences from a library of nucleic acids,proteins having different structure and/or function from a library ofproteins, drug candidates from a combinatorial library of smallmolecules, cells from a culture, tissue or organism, etc.

In some embodiments, analytes can be distributed on pads such that theyare individually resolvable. For example, a single molecule of eachanalyte can be present at each pad. Alternatively, analytes can bepresent as colonies or populations such that individual molecules orcells are not necessarily resolved. The colonies or populations can behomogenous with respect to containing only a single species of analyte(albeit in multiple copies). Taking nucleic acids as an example, eachpad in an array of pads can include a colony or population of nucleicacids and every nucleic acid in the colony or population can have thesame nucleotide sequence (either single stranded or double stranded).Colonies of nucleic acids can also be referred to as ‘nucleic acidclusters’. Nucleic acid colonies can optionally be created by clusteramplification or bridge amplification techniques as set forth in furtherdetail elsewhere herein. Multiple repeats of a target sequence can bepresent in a single nucleic acid molecule, such as a concatamer createdusing a rolling circle amplification procedure. Thus, a responsive padcan contain multiple copies of a single species of an analyte.Alternatively, a colony or population of analytes that are at a pad caninclude two or more different species. For example, one or more pads inan array of pads can each contain a mixed colony having two or moredifferent nucleic acid species (i.e. nucleic acid molecules withdifferent sequences). The two or more nucleic acid species in a mixedcolony can be present in non-negligible amounts, for example, allowingmore than one nucleic acid to be detected in the mixed colony.

As set forth above for target analyte features in general, each pad inan array of pads can include a plurality of nucleic acid clusters andeach cluster can occur in the detection zone for a single pixel.Alternatively, each cluster can occur in the detection zones for severalpixels. For example, each pad can include only a single nucleic acidcluster and the cluster can be included in the detection zones for atleast two pixels.

In some embodiments, at least two nucleic acid clusters are included inthe detection zone for a single pixel. Each of the clusters can behomogenous with respect to the nucleotide sequence present and the twoclusters can have different nucleotide sequences compared to each other.The clusters can be labeled, for example, by a labeled nucleotide thathas been incorporated in the course of a sequencing-by-synthesis (SBS)technique. Other labels are possible too such as those set forthelsewhere herein. The two clusters can have the same label as each otheror a different label can be present at each of the two clusters. Forexample, in an SBS technique the identity of the labels will depend uponthe sequences of the two clusters at the position of nucleotideincorporation. This example can be extended to formats where 3 or 4 ormore nucleic acid clusters are included in the detection zone for asingle pixel. Accordingly, multiple clusters can have differentnucleotide sequences from each other and can be labeled with probes thatare the same or different.

As used herein, the term “different”, when used in reference to nucleicacids, means that the nucleic acids have nucleotide sequences that arenot the same as each other. Two or more different nucleic acids can havenucleotide sequences that are different along their entire length.Alternatively, two or more different nucleic acids can have nucleotidesequences that are different along a substantial portion of theirlength. For example, two or more different nucleic acids can have targetnucleotide sequence portions that are different from each other whilealso having a universal sequence region that is the same for both (orall).

In particular embodiments, two clusters that are present in thedetection zone for the same pixel can be distinguished from each otherdue to the presence of different priming sequences. A first of the twoclusters can be selectively hybridized to a first primer that hasspecificity for a first priming sequence present the first clustercompared to a second priming sequence that is present on the secondcluster. The first cluster can be detected or sequenced based onhybridization of the first primer, for example, if the first primer hasa detectable label or if the first primer undergoes a reaction, such asnucleotide extension, oligonucleotide ligation, sequencing by synthesisor other technique, that recruits label(s) to the cluster. In turn, thesecond cluster can be detected based on specificity for a second primerto the second priming sequence compared to the first priming sequence.The second cluster can be selectively detected or sequenced using thetechniques exemplified above for the first cluster.

Analytes can be attached to a responsive pad. The attachment can becovalent or non-covalent. In some embodiments, the attachment can bemediated by a gel material. The analytes can be nucleic acids that areattached to a gel material. Exemplary methods and reactants forattaching nucleic acids to gels are described, for example, in US Pat.App. Pub. No. 2011/0059865 A1, or U.S. patent application Ser. No.13/784,368, each of which is incorporated herein by reference. Nucleicacids can be attached to the gel or to the surface of a pad via their 3′oxygen, 5′ oxygen, or at other locations along their length such as viaa base moiety of the 3′ terminal nucleotide, a base moiety of the 5′nucleotide, and/or one or more base moieties elsewhere in the molecule.Non-covalent modes of attachment include, for example, ionicinteractions between nucleic acid and a surface (or gel), entrapment ofnucleic acid within pores of a gel, protein-protein interactions,binding between receptors and ligands and/or nucleic acid, and otherknown modes.

An apparatus of the present disclosure can include a flow cell.Exemplary flow cells, methods for their manufacture and methods fortheir use are described in US Pat. App. Publ. Nos. 2010/0111768 A1 or2012-0270305 A1; or WO 05/065814, each of which is incorporated hereinby reference. Flow cells provide a convenient format for housing anarray of responsive pads and that is subjected to asequencing-by-synthesis (SBS) reaction or other technique that involvesrepeated delivery of reagents in cycles (e.g. synthesis techniques ordetection techniques having repetitive or cyclic steps).

Any of a variety of labels or moieties can be present at a responsivepad. Exemplary labels and moieties include, but are not limited tofluorophores, chromophores, chemiluminescent species, electrochemicalluminescence species, fluorescence quenchers, donors and/or acceptorsfor fluorescence resonance energy transfer (FRET), nanocrystals and thelike. Fluorophores that may be useful include, for example, fluorescentlanthanide complexes, including those of Europium and Terbium,fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosin,coumarin, methyl-coumarins, pyrene, Malacite green, Cy3, Cy5, stilbene,Lucifer Yellow, Cascade Blue, Texas Red, Alexa dyes, phycoerythin, andothers known in the art such as those described in Haugland, MolecularProbes Handbook, (Eugene, Oreg.) 6th Edition; The Synthegen catalog(Houston, Tex.), Lakowicz, Principles of Fluorescence Spectroscopy,2^(nd) Ed., Plenum Press New York (1999), or WO 98/59066, each of whichis hereby incorporated by reference. Exemplary quenchers include, butare not limited to, DACYL(4-(4′-dimethylaminophenylazo)benzoic acid),Black Hole Quenchers (Biosearch Technologies, Novato, Calif.), Qxlquenchers (Anaspec, Freemont, Calif.), Iowa black quenchers, DABCYL,BHQ1, BHQ2, QSY7, QSY9, QSY21, QSY35, BHQO, BHQ1, BHQ2, QXL680,ATTO540Q, ATTO580Q, ATTO612Q, DYQ660, DYQ661 and IR Dye QC-1 quenchers.Chemiluminescent species include, for example, luminal, reagents usedfor detection in pyrosequencing, aequorin and other species known in theart. Exemplary electrochemical luminescence species include, but are notlimited to Ru(bpy₃)²⁺, Bodipy dyes, luminal derivatives, acridine estersand others known in the art.

A label or moiety can be selected to suit a particular application of anapparatus, system or method set forth herein. For example, the label ormoiety can be associated with a target analyte that is present at thepad and detected using a technique set forth below. The label or moietycan be in a detectable state or a non-detectable state, for example, asinfluenced by a characteristic of a responsive pad at which the label ormoiety is present. Thus, a probe or moiety can be located in a detectionzone of a pixel and the probe or moiety can optionally be in a statethat produces a signal that is detected by the pixel.

In particular embodiments, target analytes include fluorescent moietiesand the pixels are configured to detect emission from the fluorescentmoieties. An apparatus that includes the fluorescent moieties canfurther include an excitation assembly as set forth herein or otherwiseknown in the art. An apparatus with an excitation assembly is alsouseful when using fluorescence quenchers, donors and/or acceptors forfluorescence resonance energy transfer (FRET) or nanocrystals.Responsive pads where target analytes occur can be selectively placed ina state to preferentially produce fluorescent signals. For example, aresponsive pad can be electrically activated to create an electric fieldthat selectively attracts a fluorescent label, donor, acceptor ornanocrystal to the pad, thereby producing a fluorescent signal at thepad. Alternatively, a responsive pad can be electrically activated tocreate an electric field that selectively repels or degrades afluorescence quencher, thereby preferentially producing a fluorescentsignal from a label or moiety that was previously quenched. In yetanother example, a responsive pad can be electrically activated tocreate an electric field that selectively repels or degrades afluorescent label, donor, acceptor or nanocrystal, thereby inhibitingfluorescent signals from the pad. Such inhibition can also result byelectrically activating a pad to create an electric field thatselectively attracts a fluorescence quencher. It is also possible toplace a responsive pad in a state (e.g. a neutral state) where the abovereagents are allowed to diffuse away. Differential activation ofresponsive pads in such ways can be used to achieve super resolutiondetection of fluorescent target analytes present at multiple responsivepads in the detection zone of a single pixel.

Similarly, responsive pads having chemiluminescent moieties orelectrochemical luminescence moieties can be placed in different statesto achieve super resolution imaging. An apparatus used forchemiluminescent or electrochemical luminescence detection can beconfigured similar to that exemplified above for use of fluorescentmoieties, except that an excitation assembly is not necessary. Rather,signal generation can be achieved by activating a responsive pad tocreate an electric field that selectively attracts a chemical capable ofgenerating chemiluminescence, and signal can be inhibited by activatingthe pad to create an electric field that repels or degrades thechemical. Electrochemical luminescence signal generation can be achievedby activating a responsive pad to carry out a redox reaction thatproduces the signal, and switching the pad can inhibit signal due to thepad being in a state where the redox reaction does not occur.Conversely, a responsive pad can be actuated to carry out a redoxreaction that inhibits signal and a switch can alter the pad to a statewhere the redox reaction does not occur so that signal can be generated.

Several embodiments set forth herein illustrate differentiation of twoor more features in the detection zone of the same pixel usingdifferential activation of responsive pads to which the features areattached (or otherwise functionally associated) during a detection step,between detection steps or after a detection step. Alternatively oradditionally, two features that are present in the detection zone of thesame pixel can be distinguished based on a chemical or physicaldistinction that is introduced prior to a particular detection step.Taking as an example nucleic acid features, two or more pads can bedifferentially actuated during one or more of target nucleic acidcapture, target nucleic acid amplification or hybridization of a primerused for detection of the nucleic acids. For example, a first library oftarget nucleic acids that includes members having a first universalpriming sequence can be captured on a first subset of responsive padsand/or amplified on the first subset of pads. A second library of targetnucleic acids that includes members having a second universal primingsequence can be captured on a second subset of responsive pads and/oramplified on the second subset of pads. The pixels and features can bearranged such that a feature from the first subset and a feature fromthe second subset are both located in the detection zone of a particularpixel. These two features can be distinguished without necessarilyapplying differential actuation during a detection step, after adetection step or between detection steps. Rather, the features can bedistinguished based on nucleic acid hybridization specificity whereby afirst primer selectively hybridizes to the first universal primingsequence and a second primer selectively hybridizes to the seconduniversal priming sequence.

In the above examples, it will be understood that changing the state ofresponsive pads relative to each other can result in filling a pad tocapacity with a particular reagent (e.g. label or probe), removing allof a particular reagent from a pad, degrading all of a particularreagent at a pad or modifying all of a particular reagent at a pad.However, in most embodiments it will be sufficient and in some caseseven desirable that differential actuation of responsive pads results inhigher relative concentrations of a particular reagent at one padcompared to another. For example an attractive electric field applied ata first pad can create a relatively higher concentration of afluorophores, chromophores, chemiluminescent species, electrochemicalluminescence species, fluorescence quenchers, donors and/or acceptorsfor fluorescence resonance energy transfer (FRET), nanocrystals or otherreagents at the first pad compared to at a second pad where the field isnot applied or where a different field is applied. Similarly, arepellant or destructive field applied at a first pad can create arelatively lower concentration of these or other reagents at the firstpad compared to at a second pad where the field is not applied or wherea different field is applied.

FIG. 3 shows side profile views of two detection apparatus that have anarray of pixels and an array of responsive pads configured as diagrammedin FIG. 2. Panel A shows an integrated apparatus 200 wherein the arrayof pixels and the array of responsive pads are fixed on a substrate. Thepixels are optically coupled to detection zones via light pipes in thesubstrate. For example, signal from nucleic acid clusters 3 a and 3 b indetection zone 1 a passes through light pipe 12 to reach pixel 13. Thelight pipe is bounded by vertical curtains 11 a and 11 b that preventcross talk of optical signals between pixels. Nucleic acid cluster 3 ais attached to responsive pad 2 a and cluster 3 b is attached to pad 2b. Pads 2 a and 2 b can be independently actuated to allow superresolution imaging of clusters 3 a and 3 b. As shown in Panel A, atleast a portion of each pad is in the detection zones for two pixels.For embodiments that use fluorescence detection, light pipe 12 caninclude an optical filter that blocks excitation radiation from reachingthe pixel 13. An advantage of the configuration shown in FIG. 3, Panel Ais that the array of pixels and array of responsive pads arepositionally fixed relative to each other. This design does not requirealignment and focusing devices used in multi-component designs where thetwo arrays can move relative to each other.

Panel B of FIG. 3 shows a detection apparatus 300 having a cameracomponent 15 that is separated in space from the substrate that is to bedetected 16. In such a configuration the camera component 15 can bemoved relative to the array of responsive pads that is located onsubstrate 16, for example, to achieve a desired focus, resolution oralignment. Pixel 13 is directed to substrate 16 and has a detection area1 a that includes two nucleic acid clusters 3 a and 3 b on separateresponsive pads 2 a and 2 b, respectively. The camera in Panel B isconfigured for epifluorescent detection when an excitation assembly ispresent, for example, at a position that excites the nucleic acidclusters from the same side of substrate 16 that is observed by thepixels. Again, pads 2 a and 2 b can be independently actuated to allowsuper resolution imaging of clusters 3 a and 3 b. At least a portion ofeach of the pads is in the detection zones for two pixels.

An apparatus of the present disclosure can include an activation circuitto actuate changes in the characteristics of responsive pads in an arrayof pads. FIG. 4 shows a diagrammatic representation of a detectionapparatus 100 including a detection module 10 (including an array ofreactive pads 2 and an array of pixel detection areas 1), an activationcircuit 20 configured to actuate changes in each of the responsive pads2 and, optionally, to receive feedback regarding the state of each pad2. As set forth previously herein, the activation circuit can beconfigured to individually address and actuate each of the responsivepads. The activation circuit can produce changes in any of a variety ofcharacteristics at or near the responsive pads including, but notlimited to, presence or absence of electric charge; positive, negativeor neutral polarity of electric charge; strength of electric field;shape of electric field; presence or absence of electric current;direction of electric current; strength of electric current; type ofelectric current (e.g. DC and/or AC); shape for AC current waveform;frequency or magnitude of AC current; magnetic state; presence orabsence of a magnetic dipole; chemical properties at the surface such aspH, redox potential, hydrophobicity, hydrophilicity, presence of areactive species or absence of a reactive species; and the like.

Thus, an activation circuit can place responsive pads into differentstates. As used herein, the term “different” or “differential,” whenused in reference to a responsive pad, means that the pad has at leastone characteristic that is absent at another pad or that is not presentto the same degree as at another pad. The characteristic is typicallyresponsive to an activation circuit or other device that actuates changeat the pad. For example, pads can be different with respect to changesin the characteristics set forth herein, for example, with regard to anactivation circuit. It will be understood that the phrase “a differentelectric field” can be used to refer to the presence or absence of anelectric field, such that a pad that has no electric field can beconsidered to have a different electric field from a pad that has anelectric field unless explicitly stated to the contrary. Other differentstates include, for example, changes in hydrophobicity andhydrophilicity. Such changes in state can occur due to electrowettingand other techniques known in the art for manipulating droplets examplesof which are set forth in US Pat. App. Pub. No. 2013/0116128 A1, whichis incorporated herein by reference.

In particular embodiments an activation circuit is configured to applyan electric field at first and second pads in an array of pads (forexample at two pads in the detection zone of a single pixel). Theactivation circuit can be configured to apply a different electric fieldat the first pad compared to the second pad, and the activation circuitcan have a switch to selectively alter the electric field at the firstpad compared to the second pad.

As used herein, the term “selectively alter” means to alter one thing(e.g. a first pad) to a greater degree than another thing (e.g. a secondpad). In some cases selective alteration can be achieved by turning onething on and another thing off. However, it is also possible to make aselective alteration without turning one of the things on and insteadreducing or increasing an actuatable characteristic of one of the thingsrelative to the other thing. Selective alteration can result in changesfor any of the characteristics set forth herein, for example, above withregard to an activation circuit. For example, an activation circuit canbe configured to apply (or relatively increase) electric field at afirst pad while preventing (or relatively decreasing) electric field ata second pad, and a switch can be configured to prevent (or relativelydecrease) electric field at the first pad while applying (or relativelyincreasing) electric field at the second pad. Similarly, an activationcircuit can be configured to apply positive electric field at a firstpad while applying negative electric field at a second pad, and a switchcan be configured to apply negative electric field at the first padwhile applying positive electric field at the second pad. As a furtherexample, a pad can be actuated to attract or repel an aqueous droplet orthe contents of an aqueous droplet. In particular embodiments a pad isactuated by electrowetting to attract or repel a droplet.

An apparatus of the present disclosure can further include a readoutcircuit to acquire signals from an array of pixels. The detectionapparatus 100 diagrammed in FIG. 4 includes a detection module 10(including an array of reactive pads 2 and an array of pixel detectionareas 1), an activation circuit 20 and a readout circuit 30. The readoutcircuit 30 can be configured to obtain signal information from the pixeldetection areas 1 via the array of pixels. The readout circuit canoptionally be configured to alter the gain of individual pixels or toturn pixels on and off in response to the amount of signal received byone or more pixels in the array of pixels.

An apparatus of the present disclosure can be included in a detectionsystem 500 as diagrammed in FIG. 4. The system can include detectionapparatus 100 fluidically coupled to a fluidic system 70 and inoperative communication with a control module 50 and a processing module60. The fluidic system 70 can be configured to deliver fluid reagentsused in a detection method that occurs in the detection apparatus 10. Insome embodiments, such as those using sequencing-by-synthesis as setforth below, the fluidic system can deliver reagents in repeated cycles.Repeated cycles of fluid delivery can also be useful for otherapplications where polymeric molecules are synthesized or sequenced, orfor other applications. Alternatively, a fluidic system can beconfigured for non-cyclic delivery of reagents to a given array of padsor at least for a given sample present on the array of pads. Examples offluid systems that are coupled to biological arrays and that can bereadily adapted to deliver fluids to an array of pads set forth hereinare described in US Pat. App. Pub. No. US 2010/0009871 A1; US Pat. App.Pub. No. 2012/0270305 A1 and U.S. patent application Ser. No.13/766,413, (published as US Pat. App. No. 2013/0260372 A1) each ofwhich is incorporated herein by reference. Particularly useful fluidicsystems are those that move fluid droplets to and from detection areasvia electrowetting or other techniques as described, for example, inU.S. patent application Ser. No. 13/670,318 (published as US Pat. App.Pub. No. 2013/0116128 A1), which is incorporated herein by reference.

The control module 50 can be configured to direct the readout circuit 30to acquire signals from each of the pixel detection areas 1 during asensing period. The control module 50 can also communicate with theactivation circuit 20 to direct actuation of responsive pads during thesensing period. The communication from the control module 50 can directthe activation circuit to switch the actuation at the responsive pads.In an exemplary embodiment of the system diagrammed in FIG. 4, thecontrol module 50 can direct the readout circuit 30 to acquire signalsfrom each of the pixels during a sensing period, direct the activationcircuit 20 to apply a different electric field at the first pad comparedto the second pad, during the sensing period, and direct the activationcircuit to switch to selectively alter the electric field at the firstpad compared to the second pad, during the sensing period.

As used herein, the term “sensing period” means a time frame, whethercontinuous or discontinuous, during which signal is collected.Accordingly, a control module can direct a readout circuit to acquiresignals from a pixel continuously during a sensing period, oralternatively, the control module can direct the readout circuit totoggle the pixel between an on-state and an off-state during a sensingperiod. Similarly, the gain at a pixel can be increased or decreasedduring a sensing period.

Control module 50 can be further configured to communicate with thefluidic system 70. The control module 50 can provide instructions to thefluidic system to direct reagent delivery from a particular reservoir,or other fluidic component, to the detection apparatus 100. Whenmultiple fluids are to be delivered, for example as is the case for asequencing-by-synthesis protocol, the control module 50 can direct thesequence of fluid components delivered to the detection apparatus 100.Generally, control module 50 can also direct the amount of fluiddelivered at a particular step of a protocol, the duration of deliveryof a particular fluid, the temperature for a particular fluid, the rateof fluid delivery (e.g. via changes in fluid pressure), and the like.The control module 50, being in communication with the fluidic system70, the readout circuit 30, the detection module 10, and the activationcircuit 20 can coordinate super resolution detection of a plurality oftarget analytes that are chemically manipulated in an array-basedplatform. An example of such a platform is one using asequencing-by-synthesis technique.

In particular embodiments, the control module 50 can also be configuredto receive feedback from the activation circuit 20, readout circuit 30,fluidic system 70 or detection module 10. Feedback from one or more ofthese components can be used to modify directions sent to thecomponent(s) from which feedback was received or another component ofthe system. Thus, the control module can assess the overall condition ofthe system and modify function to achieve desired output or activity.Exemplary algorithms and configurations for assessing and modifyingfunction of an array-based detection system are described in U.S. Pat.No. 8,244,479, which is incorporated herein by reference. In particularembodiments, the control module 50 can also be in communication with theprocessing module 60, to send directions or receive feedback pertainingto the functions of the processing module 60.

A processing module 60 that is included in a system 500 of the presentdisclosure can be in communication with a readout circuit 30. Theprocessing module 60 can receive signals from the readout circuit 30 andmodify the signals to create data in a desired format. Taking an SBSsystem as an example, the processing module 60 can determine theidentity of a nucleotide that is incorporated at a particular nucleicacid cluster from electrical signals obtained by a pixel during asensing period and from a schedule of actuation periods for two or morepads that were in the detection zone of the pixel during the sensingperiod. The processing module 60 can further include algorithms tomanipulate data to provide a desired output that can be communicated toa user. For example, data can be used to determine presence or absenceof a target analyte (e.g. presence of a nucleic acid sequence orpresence of a single nucleotide polymorphism in a sequence), amount of atarget analyte (e.g. ploidy level for a gene sequence or expressionlevel for an RNA sequence), structure of a target analyte (e.g.nucleotide sequence of a nucleic acid), chemical reactivity of a targetanalyte (e.g. binding affinity between receptor and ligand or kineticsof reaction for an enzyme) or the like.

Processing module 60 can also be configured to send directions to othercomponents of the system based on data obtained or processed. Forexample, processing module 60 can determine when enough information hasbeen obtained from a sample that further manipulation and/or observationof the sample can be stopped. Directions can then be sent to thedetection apparatus, for example via the control module, to cease orpause data acquisition.

The various components of system 500 or other system of the presentdisclosure can be present in a single unit, for example, having arelatively small footprint. Alternatively, the components can bedistributed, for example, in a network that includes data connectionsand in some cases fluidic connections. In some embodiments, informationprocessing modules can be distributed in a computer network that isconnected to other components of the system. In some cases one or moreof the processing modules can be cloud-based. Exemplary, cloud-basedsystems for processing sequencing data and that can be adapted for usein a system or method of the present disclosure are described in U.S.patent application Ser. No. 13/790,596 (published as US Pat. App. Pub.No. 2013/0275486 A1) and Ser. No. 13/790,623 (published as US Pat. App.Pub. No. 2013/0274148 A1), each of which is incorporated herein byreference.

A system provided by the present disclosure can be used for sequencingnucleic acids. The system can include (a) a detection apparatus having(i) an array of electrically responsive pads on a substrate surface;(ii) an array of pixels, wherein each pixel in the array has a detectionzone on the surface that includes a subset of four of the pads; and(iii) an activation circuit to apply an electric field to the pads inthe subset individually, wherein the activation circuit is configured toapply a different electric field at a first pad of the subset comparedto the other pads of the subset; (b) a readout circuit to acquiresignals from the array of pixels; (c) a control module that directs thereadout circuit to acquire signals from each of the pixels during asensing period and that directs the activation circuit to sequentiallyapply different electric fields at the four pads during the sensingperiod; and (d) a processing module that correlates (i) the signalsacquired from the pixels during the sensing period and (ii) thesequential application of the different electric fields at the four padsduring the sensing period, in order to distinguish a sequence of signalsfor each of the pads.

The control module and processing module of a nucleic acid sequencingsystem can be configured to carry out a sequencing-by-synthesis (SBS)protocol. In SBS, extension of a nucleic acid primer along a nucleicacid template (e.g. a target nucleic acid or amplicon thereof) ismonitored to determine the sequence of nucleotides in the template. Theunderlying chemical process can be polymerization (e.g. as catalyzed bya polymerase enzyme). In a particular polymerase-based SBS embodiment,fluorescently labeled nucleotides are added to a primer (therebyextending the primer) in a template dependent fashion such thatdetection of the order and type of nucleotides added to the primer canbe used to determine the sequence of the template. A plurality ofdifferent templates at different features on an array of responsive padsset forth herein can be subjected to an SBS technique under conditionswhere events occurring for different templates can be distinguishedusing super resolution imaging.

Flow cells provide a convenient format for housing an array of nucleicacid clusters located on responsive pads that are subjected to an SBStechnique that involves repeated delivery of reagents in cycles.Exemplary flow cells are set forth above and in references cited above.To initiate a first SBS cycle, one or more labeled nucleotides, DNApolymerase, etc., can be flowed into/through a flow cell that houses anarray of nucleic acid clusters that have been hybridized to a sequencingprimer. Those sites of an array where primer extension causes a labelednucleotide to be incorporated can be detected. Optionally, thenucleotides can further include a reversible termination property thatterminates further primer extension once a nucleotide has been added toa primer. For example, the labeled nucleotide that is contacted with thenucleic acid clusters can have a reversible terminator moiety that getsadded to a primer such that subsequent extension cannot occur until adeblocking agent is delivered to remove the moiety. Thus, forembodiments that use reversible termination, a deblocking reagent can bedelivered to the flow cell (before or after detection occurs). Washescan be carried out between the various delivery steps. The cycle canthen be repeated n times to extend the primer by n nucleotides, therebydetecting a sequence of length n. Exemplary SBS procedures, fluidicsystems and detection system components that can be readily adapted foruse in a system of method of the present disclosure are described, forexample, in Bentley et al., Nature 456:53-59 (2008), WO 04/018497; U.S.Pat. No. 7,057,026; WO 91/06678; WO 07/123744; U.S. Pat. Nos. 7,329,492;7,211,414; 7,315,019; 7,405,281, and US 2008/0108082, each of which isincorporated herein by reference.

Other sequencing procedures that use cyclic reactions can be used, suchas pyrosequencing. Pyrosequencing detects the release of inorganicpyrophosphate (PPi) as particular nucleotides are incorporated into anascent nucleic acid strand (Ronaghi, et al., Analytical Biochemistry242(1), 84-9 (1996); Ronaghi, Genome Res. 11(1), 3-11 (2001); Ronaghi etal. Science 281(5375), 363 (1998); U.S. Pat. Nos. 6,210,891; 6,258,568and U.S. Pat. No. 6,274,320, each of which is incorporated herein byreference). In pyrosequencing, released PPi can be detected by beingimmediately converted to adenosine triphosphate (ATP) by ATPsulfurylase, and the level of ATP generated can be detected vialuciferase-produced photons. Thus, the sequencing reaction can bemonitored by detecting photons released during this chemiluminescentreaction. Accordingly, excitation radiation sources used forfluorescence based detection systems are not necessary forpyrosequencing procedures. Useful fluidic systems, detectors andprocedures that can be used for application of pyrosequencing to arraysof the present disclosure are described, for example, in WIPO Pat. App.Ser. No. PCT/US11/57111, US 2005/0191698 A1, U.S. Pat. Nos. 7,595,883,and 7,244,559, each of which is incorporated herein by reference.

Sequencing-by-ligation reactions are also useful including, for example,those described in Shendure et al. Science 309:1728-1732 (2005); U.S.Pat. Nos. 5,599,675; and 5,750,341, each of which is incorporated hereinby reference. Some embodiments can include sequencing-by-hybridizationprocedures as described, for example, in Bains et al., Journal ofTheoretical Biology 135(3), 303-7 (1988); Drmanac et al., NatureBiotechnology 16, 54-58 (1998); Fodor et al., Science 251(4995), 767-773(1995); and WO 1989/10977, each of which is incorporated herein byreference. In both sequencing-by-ligation andsequencing-by-hybridization procedures, target nucleic acids (oramplicons thereof) that are present at sites of an array are subjectedto repeated cycles of oligonucleotide delivery and detection. Typically,the oligonucleotides are fluorescently labeled and can be detected usingfluorescence detectors similar to those described with regard to SBSprocedures herein or in references cited herein.

A fluidic system used in a system or method set forth herein can storeand deliver one or more of the reagents or fluid components set forthabove in the context of target nucleic acid capture, amplification ornucleotide sequencing protocols. For example, the reagents can be storedin appropriate reservoirs prior to delivery to a flow cell or substratesuch as one already having an array of nucleic acids. Furthermore, adetection apparatus can include a nucleic acid feature (e.g. a nucleicacid cluster) having an intermediate species produced by one of thesteps (e.g. a primer attached to a labeled and reversibly terminatednucleotide, or a primer attached to a labeled nucleotide that lacks aterminator, or a nucleic acid attached to a polymerase and/ornucleotide). The nucleic acid feature can in turn be attached to aresponsive pad that is in one of the states exemplified herein forproducing a signal to be detected or in a state for inhibiting a signalfrom being detected at the nucleic acid feature. Different features canhave differential properties such as different priming sites ordifferent primers hybridized to the features.

A processing module 60 that is included in a sequencing system can beconfigured to correlate (i) the signals acquired from the pixels duringthe sensing period, (ii) the sequential activation of different padsduring the sensing period, and (iii) the sequence of reagents deliveredto the substrate in order to distinguish a sequence of signals for eachof the pads and to distinguish a sequence of reagents that produce thesignals at each of the pads. The nucleotide sequence of nucleic acidfeatures at each pad can in turn be determined from this correlation.The nucleotide sequences or the signal data can be exported to anotherprocessing device for sequence analysis.

The present disclosure also provides a detection apparatus that includes(a) an array of responsive pads on a substrate surface, wherein eachresponsive pad includes a nucleic acid feature of a plurality of nucleicacid features in the array, wherein a first subset of nucleic acidfeatures in the plurality of nucleic acid features have a firstuniversal sequence and different target sequences, wherein a secondsubset of nucleic acid features in the plurality of nucleic acidfeatures have a second universal sequence and different targetsequences, wherein the first universal sequence is different from thesecond universal sequence; (b) an array of pixels, wherein each pixel inthe array has a detection zone on the surface that includes at least twonucleic acid features of the plurality of nucleic acid features, the atleast two nucleic acid features including a nucleic acid from the firstsubset of nucleic acid features and a nucleic acid from the secondsubset of nucleic acid features; and (c) an activation module to alter acharacteristic of a pad in the first subset and of a pad in the secondsubset, wherein the activation module is configured to apply a differentcharacteristic at the pad in the first subset compared to the pad in thesecond subset, and wherein the activation module has a switch toselectively alter the characteristic at the pads in the first and secondsubsets.

Optionally, the detection apparatus can be included in a nucleic acidsequencing system that also includes (I) a readout circuit to acquiresignals from the array of pixels; (II) a control module that directs thereadout circuit to acquire signals from each of the pixels during asensing period and that optionally directs the activation circuit tosequentially actuate different responsive pads in each of the detectionzones during the sensing period; and (III) a processing module thatoptionally correlates (i) the signals acquired from the pixels duringthe sensing period and (ii) the sequential actuation of the differentresponsive pads during the sensing period, in order to distinguish asequence of signals for each of the pads.

The present disclosure further provides a method of detecting analytes.The method can include the steps of (a) providing a detection apparatushaving an array of electrically responsive pads and an array of pixels,wherein each pixel in the array has a detection zone that includes asubset of at least two of the electrically responsive pads, wherein thetwo pads include different target analytes, respectively; (b) acquiringsignals from each of the pixels while selectively applying an electricfield at a first of the two pads to preferentially produce signal from afirst of the different target analytes compared to a second of thetarget analytes, thereby preferentially acquiring signals from the firstof the target analytes compared to the second of the target analytes;and (c) acquiring signals from each of the pixels while selectivelyapplying an electric field at the second of the two pads topreferentially produce signal from the second of the different targetanalytes compared to the first of the target analytes, therebypreferentially acquiring signals from the second of the target analytescompared to the first of the target analytes.

As used herein, the term “preferentially acquire,” when used inreference to a signal, means to detect more signal from one source (e.g.from a first target analyte at a first pad) than from another source(e.g. a second target at a second pad). In some cases preferentialacquisition can be achieved by detecting signal only from one source andnot from another. However, it is also possible to detect more of asignal or a different type of a signal, from one source compared toanother when preferentially acquiring signal.

A method set forth herein can be carried out using an apparatus orsystem exemplified herein. However, other suitable apparatus or systemscan be used as desired for a particular application of the methods. Inparticular embodiments, a detection method can include a step ofacquiring signals from a pixel while selectively actuating a first of atleast two pads that are in the detection zone of the pixel. This canallow signal to be preferentially produced from a first target analytethat is at one of the pads compared to a second target analyte that isat the other pad. Thus, signals can be preferentially acquired from thefirst target analytes even when the second target analytes are in thefield of view of the pixel.

In one exemplary embodiment, the target analytes can produce signal whenthe pads to which they are attached are actuated to produce an electricfield. This can be demonstrated for a pad that attracts a label to thetarget analyte when the electric field is turned on. In this case, atarget analyte that is present at a pad that is not activated to producethe electric field (or at a pad that produces a substantially weakerfield or at a pad that is activated to produce a field of oppositepolarity) will not produce signal and will not be detected. The electricfields at the two pads can be switched to change the pad from whichsignal is detected by the pixel. As such, acquiring signal from thepixel and accounting for the schedule of states for the pads can be usedto achieve super resolution detection of the different target analytesthat are simultaneously in the detection zone of the pixel. Thus,different target analytes that are simultaneously present in thedetection zone of a single pixel can be distinguished by selectivespatial activation (or inhibition) of signal generation duringdetection. Any of a variety of labels and detectable moietiesexemplified elsewhere herein can be used similarly.

In an alternative embodiment, the target analytes can be prevented orinhibited from producing signal when the pads to which they are attachedare actuated to produce an electric field. This can be demonstrated fora pad that has a label present and that attracts a quencher when theelectric field is turned on. FIG. 5 provides a diagrammaticrepresentation of a detection apparatus in three different states. In afirst state (Panel A) neither of the two pads is charged and thequencher (e.g. black hole quencher) is not attracted to fluorescentlylabeled clusters that are present at either of the pads. Thus, clustersat both pads are fully capable of producing fluorescent signals. Panel Bshows the state where the pad at the left side of each detection zone ispositively charged, thereby creating an electric field that attracts thenegatively charged black hole quencher to the cluster. As such, thenucleic acid clusters at the left side pads will be quenched and thepixel will detect little to no signal from these clusters. However, theright side pads will produce “unquenched” signal that is detected by thepixel. As demonstrated by Panel C, the electric fields at the two padscan be switched to change the cluster from which signal is detected byeach pixel, thereby allowing super resolution imaging. In this case, thepads on the right attract quencher, decreasing or preventing signaldetection, and the pads on the left release quencher to producefluorescent signal that is detectable by the pixel. Again, any of avariety of labels and detectable moieties exemplified elsewhere hereincan be used similarly.

Further by way of example, selectively applying an electric field at thefirst of two pads in a method of the present disclosure can attract afluorescence quencher to the first pad to preferentially quenchfluorescence at the first pad, thereby preferentially producing signalat the second of the two pads. In this example a switch can be used toselectively apply the electric field at the second pad to attract thefluorescence quencher to the second pad to preferentially quenchfluorescence at the second pad, thereby preferentially producing signalat the first pad.

In a second example, selectively applying an electric field at the firstpad in a method of the present disclosure can attract a fluorescentlabel to the first pad to preferentially produce fluorescence at thefirst pad compared to the second pad. In this second example, a switchcan be used to selectively apply the electric field at the second pad toattract the fluorescent label to the second pad to preferentiallyproduce fluorescence at the second pad compared to the first pad.

In a third example, selectively applying an electric field at the firstpad can induce luminescence from electrochemical luminescence labels atthe first pad to preferentially produce luminescence at the first padcompared to the second pad. In this third example, a switch can be usedto selectively apply the electric field at the second pad to induceluminescence from electrochemical luminescence labels at the second padto preferentially produce luminescence at the second pad pads comparedto the first pad.

Selective activation that allows super-resolution imaging need not occurduring a detection step or even during a sensing period. For example,prior to the sensing period of a solid-phase SBS protocol, subsets oftarget nucleic acids in a library can be selectively captured at pads ofa solid support, selectively amplified to form features on pads of asolid support or selectively hybridized to an SBS primer.

Accordingly, provided herein is a method of detecting target nucleicacids, including the steps of (a) providing a substrate comprising anarray of pads, the array of pads including a first subset of the padsand a second subset of the pads; (b) delivering a first solution to thesubstrate, wherein the first solution includes a first plurality ofdifferent target nucleic acids that selectively attach to the firstsubset of pads compared to the second subset of pads; (c) delivering asecond solution to the substrate, wherein the second solution includes asecond plurality of different target nucleic acids that selectivelyattach to the second subset of pads compared to the first subset ofpads; and (d) detecting the substrate using an apparatus having an arrayof pixels, wherein each pixel in the array has a detection zone thatincludes (i) at least one of the target nucleic acids that is attachedto a pad of the first subset of pads, and (ii) at least one of thetarget nucleic acids that is attached to a pad of the second subset ofpads.

For embodiments that include delivery of a first solution and a secondsolution to a substrate, it will be understood that the solutions can bedelivered simultaneously as a preformed single solution or they can bedelivered in a way that the solutions mix to form a single solution inthe presence of the substrate. Alternatively, the first and secondsolutions can be separate solutions that are delivered to a substratesequentially. The first solution can be removed from contact with thesubstrate prior to delivery of the second solution. Optionally, thesubstrate can be treated with one or more wash solutions betweendelivery of the first solution and delivery of the second solution.

A first plurality of nucleic acids and second plurality of nucleic acidscan be differentially captured at two or more pads on a substrate basedon differential properties of the nucleic acids, differential propertiesof the pads or both. An exemplary property of the nucleic acids that canbe exploited for differential capture include, but are not limited to,presence of particular sequence regions (e.g. a priming sequence,capture sequence, or the like). These sequence regions can facilitatecapture via hybridization to complementary sequences present on captureprobes that are located at each pad. Other exemplary properties include,but are not limited to, presence of a binding moiety (e.g. a ligand orreceptor), charge, mass, length (i.e. number of nucleotides in thesequence), secondary structure (e.g. presence of single stranded ordouble stranded domains), or the like. Differential properties of thepads can include actuated or activated properties such as those setforth elsewhere herein, the sequence of attached nucleic acid captureprobes, presence or absence of nucleic acid adapters, presence orabsence of a ligand or receptor, etc.

In some embodiments, the first plurality of nucleic acids may have afirst universal sequence that is different from a second universalsequence that is present in the nucleic acids of the second plurality ofnucleic acids. Subsets of pads having capture probes that arecomplementary to the respective universal sequences will providedifferential capture.

Alternatively, subsets of unique capture probes need not be used.Rather, differential capture can be achieved by actuating a particularsubset of pads for capture in the presence of a first solution ofnucleic acids and against capture in the presence of a second solutionof nucleic acids. Thus, selective capture can be achieved by sequentialtreatment of an array of responsive pads with different nucleic acidsolutions under controlled actuation of the pads.

In a further embodiment, differential capture of target nucleic acidscan be achieved by differential pretreatment of an array of responsivepads prior to contacting the array with target nucleic acids. Anexemplary pretreatment is the modification of different responsive padsto attach adapter nucleic acids. This can be achieved by actuating aparticular subset of pads for modification in the presence of a firstsolution of adapters and against modification in the presence of asecond solution of adapters. Thus, selective modification can beachieved by sequential treatment of an array of responsive pads withdifferent adapter solutions under controlled actuation of the pads. Theadapters can be, for example, nucleic acids having a first sequenceregion that is complementary to capture probes on the pads and a secondsequence region that is complementary to a universal sequence on aparticular population of target nucleic acids. Thus, in the exampleabove, the adapters in the first solution can have a capture probecomplement sequence and a first universal sequence complement sequencewhile the adapters in the second solution have the same capture probecomplement sequence and a second universal sequence complement.Following differential treatment of the responsive pads to formadapter-modified pads, the adapter modified pads can be treated with afirst and second plurality of target nucleic acids having first andsecond universal sequences, respectively, to thereby achievedifferential capture of the target nucleic acids.

Exemplary reagents and techniques for modifying surfaces with adaptersand using the modified surfaces to attach target nucleic acids are setforth in U.S. Pat. App. No. 61/928,368, which is incorporated herein byreference. Such reagents and methods can be used in a method orcomposition set forth herein.

Although hybridization based capture is exemplified above, it will beunderstood that capture can be mediated by other physical and chemicalinteractions between nucleic acids and pads including, but not limitedto, receptor-ligand interactions, chemical crosslinking, covalent bonds,ionic interactions, magnetic interactions (e.g. Nucleic acids can beattached to beads that are held to pads via magnetism), or the like.

An array of features having two different features in the detection zoneof the same pixel, and wherein the different features have nucleic acidswith different universal sequences, can be detected by sequentially (a)hybridizing first primers to the first universal sequence of the targetnucleic acids that are attached to a first subset of pads; (b) extendingthe first primers by addition of at least one nucleotide; (c)hybridizing second primers to the second universal sequence of thetarget nucleic acids that are attached to a second subset of pads; and(d) extending the second primers by addition of at least one nucleotide,whereby signals are detected from the target nucleic acids that areattached to the first subset of responsive pads at a different time thanwhen signals are detected from the target nucleic acids that areattached to the second subset of responsive pads.

Optionally, a method of the present disclosure can include a step ofamplifying first target nucleic acids that are attached to pads of afirst subset of pads using primers that are complementary to a firstuniversal sequence that is present on the first target nucleic acids,and amplifying second target nucleic acids that are attached to pads ofthe second subset of pads using primers that are complementary to asecond universal sequence that is present on the second target nucleicacids. Amplification can be carried out by solid phase amplificationmethods set forth herein such as bridge amplification or solid-phasePCR. Accordingly, amplification can be selectively achieved when thefirst and second universal sequences are complementary to differentamplifications primers.

Selective amplification at different features can be facilitated by thepresence of different priming sequences at each feature, for example asset forth above, and/or by selective actuation of pads to attract orrepel reagents used for amplification. For example, selective actuationcan be used to selectively hybridize amplification primers to targetnucleic acids that are attached to a first subset of responsive padscompared to target nucleic acids at other pads. A method set forthherein can include a step of contacting a solution of firstamplification primers with a substrate while selectively actuating afirst subset of responsive pads, wherein the first amplification primersselectively hybridize to a first plurality of different target nucleicacids attached to the first subset of responsive pads compared to asecond plurality of different target nucleic acids attached to a secondsubset of pads. Continuing with the example, the method can optionallyinclude a step of contacting a solution of second amplification primerswith the substrate while selectively actuating the second subset ofresponsive pads in the array, wherein the second amplification primersselectively hybridize to the second plurality of different targetnucleic acids compared to the first plurality of different targetnucleic acids. The first amplification primers can have the samesequence as the second amplification primers. Alternatively, the firstamplification primers can have a different sequence compared to thesecond amplification primers.

Selective amplification can facilitate selective detection when a firsttarget nucleic acid is selectively amplified to allow detection in thezone of a pixel followed by selective amplification of the a secondtarget nucleic acid that is detected by the same pixel in the samedetection zone.

In particular embodiments, a method of detecting nucleic acids caninclude the steps of (a) providing a substrate comprising an array ofpads, the array of pads including a first subset of the pads and asecond subset of the pads; (b) contacting a first solution with thesubstrate while selectively actuating the first subset of responsivepads in the array, wherein the first plurality of different targetnucleic acids attach to responsive pads of the first subset that areselectively actuated; (c) contacting a second solution with thesubstrate while selectively actuating the second subset of responsivepads in the array, wherein the second plurality of different targetnucleic acids attach to responsive pads of the second subset that areselectively actuated; and (d) detecting the substrate using an apparatushaving an array of pixels, wherein each pixel in the array has adetection zone that includes (i) at least one of the target nucleicacids that is attached to a pad of the first subset of pads, and (ii) atleast one of the target nucleic acids that is attached to a pad of thesecond subset of pads. Optionally, the target nucleic acids include auniversal sequence that is the same for nucleic acids of the first andsecond plurality. The universal sequence of the target nucleic acidscan, in some embodiments, hybridize to capture probes attached to thefirst and second subset of pads.

In some embodiments, a method of detecting nucleic acids can include thesteps of (a) providing a substrate comprising an array of pads, thearray of pads including a first subset of the pads and a second subsetof the pads; (b) contacting a solution of adapter nucleic acids with thesubstrate while selectively actuating one or both of the subsets ofresponsive pads, wherein the adapter nucleic acids attach to responsivepads of the one or both subsets that are selectively actuated; (c)contacting a first solution with the substrate, wherein the firstplurality of different target nucleic acids attach to responsive pads ofthe first subset; (d) contacting a second solution with the substrate,wherein the second plurality of different target nucleic acids attach toresponsive pads of the second subset, wherein the responsive pads of oneor both of the first and second subset are attached to the adapternucleic acids; and (e) detecting the substrate using an apparatus havingan array of pixels, wherein each pixel in the array has a detection zonethat includes (i) at least one of the target nucleic acids that isattached to a pad of the first subset of pads, and (ii) at least one ofthe target nucleic acids that is attached to a pad of the second subsetof pads.

One or more of the adapters used in a method set forth herein caninclude a universal sequence complement that is in turn useful forhybridizing to a universal sequence of one or more target nucleic acids.Thus, an adapter can serve to mediate capture of target nucleic acids todesired pads or features of an array set forth herein. Taking theembodiment set forth above as an example, the target nucleic acids inthe first plurality can have a first universal sequence and the targetnucleic acids in the second plurality can have a second universalsequence that is different from the first universal sequence. The firsttarget nucleic acids can be selectively attached to the first subset ofpads due to the first universal sequences hybridizing to the firstadapter nucleic acids that are attached to the first subset of pads.Similarly, the selective attachment of the second target nucleic acidsto the second subset of pads can involve the second universal sequenceshybridizing to the second adapter nucleic acids that are attached to thesecond subset of pads, wherein the first adapter nucleic acids have adifferent sequence from the second adapter nucleic acids.

As set forth previously herein, a method of the present disclosure canbe used to determine the nucleotide sequences of a plurality ofdifferent nucleic acids. Another useful application is gene expressionanalysis. Gene expression can be detected or quantified using RNAsequencing techniques, such as those, referred to as digital RNAsequencing. RNA sequencing techniques can be carried out usingsequencing methodologies known in the art such as those set forth above.Gene expression can also be detected or quantified using hybridizationtechniques carried out by direct hybridization to an array of probefeatures, for example, on pads in an apparatus set forth herein. Themethods set forth herein can also be used to determine genotypes for agenomic DNA sample from one or more individual. Exemplary assays forarray-based expression and genotyping analysis that can be modified foruse in a method set forth herein are described in U.S. Pat. Nos.7,582,420; 6,890,741; 6,913,884 or 6,355,431 or US Pat. Pub. Nos.2005/0053980 A1; 2009/0186349 A1 or US 2005/0181440 A1, each of which isincorporated herein by reference.

Example I Fluorescence Quenching Using Direct Electrode Energy Transfer

This example shows that selectively applying an electric field at anelectrically responsive pad can quench fluorescent labels or moieties atthe pad. To achieve super resolution imaging, a switch can be used toalternately apply electric field at two pads that are in the detectionzone of a single pixel. Application of the field can quenchfluorescence, for example, via energy transfer between a dipole emitter(e.g. a fluorophore) and a metallic surface. A fluorescently labelednucleic acid molecule is particularly useful due to the flexibility inthe molecule which allows the fluorophore to be pulled closer to thesurface (by the field) where quenching is greatest. Other flexibletarget analytes or those having flexible linkers can be used similarly.Nucleic acids or other fluorescently labeled target analytes can beattached to a surface and surface quenching can be carried out, forexample, as described in Rant et al. Nano Lett. 4:2441-2445 (2004),which is incorporated herein by reference.

As an alternative to direct surface attachment, a nucleic acid or othertarget analyte can be attached to a gel material that is present at anelectrically responsive pad. Exemplary methods and reactants forattaching nucleic acids to gels are described, for example, in US Pat.App. Pub. No. 2011/0059865 A1, or U.S. patent application Ser. No.13/784,368, each of which is incorporated herein by reference.

FIG. 6 shows fluorescence intensity modulation after applying multiplecycles of +/−0.4 V on an electrically responsive pad coated with silanefree acrylamide that was grafted to P5 and P7 primers (SFA and P5/P7primers are described in US Pat. App. Pub. No. 2011/0059865 A1, which isincorporated herein by reference). The P5 and P7 primers were hybridizedwith HEX dye labeled fluorescent complementary primers. The voltage waspulsed every 2 sec for 0.5 sec and the intensity dipped when the voltagewas applied. The intensity modulations appear to be due to fluorescencequenching of the HEX dye label at the electrode surface. In contrast aninactive electrode did not show the intensity fluctuations influorescence signal.

Example II Fluorescence Quenching Using Energy Transfer to ElectricallyConductive Polymers

This example shows that applying an electric field at an electricallyresponsive pad that contains electrically conductive polymers in a gelcan quench fluorescent labels or moieties that are in the gel.

Transparent conductive polymers, such aspoly(3,4-ethylenedioxythiophene) poly(styrenesulfonate) (PEDOT:PSS),polyacetylenes or polyphenylenes, can be embedded in a gel, such as SFA,during the polymerization process. The gel-polymer mix can bepolymerized on the responsive pads to provide a porous electrode usingconditions previously described for polymerization of SFA (see, US Pat.App. Pub. No. 2011/0059865 A1, which is incorporated herein byreference). A target analyte such as fluorescently labeled nucleic acidcan be embedded in the gel. For example, DNA clusters can be grown inthe gel-polymer matrix using methods set forth previously herein.Application of an electric field can quench fluorescence, for example,via energy transfer between the fluorophore and the electricallyconductive polymers. The energy transfer to the fluorophores can beenhanced, compared to the surface quenching methods of Example I, due togreater proximity between the fluorophores and the conductive polymer.This can in turn lead to more efficient quenching in a conductivepolymer-gel compared to on a conductive surface.

To achieve super resolution imaging, a switch can be used to alternatelyapply electric field at two pads that are in the detection zone of asingle pixel. Pads having a fluorescently labeled target analytes in thepresence of conductive polymer-gel will be alternately quenched allowingthe target analytes to be distinguished by a single pixel having adetection zone that includes the two pads.

Example III Fluorescence Intensity Modulation with Voltage SensitiveDyes

This example shows applying an electric field at an electricallyresponsive pad that contains a target analyte (e.g. a nucleic acid)labeled with a voltage sensitive dye. Applying an electric field canmodulate signal from the dye.

An electrically responsive pad is coated with SFA that is grafted to P5and P7 primers as described in Example I. The P5/P7 primers arehybridized with complementary primers labeled with voltage sensitivedyes such as di-8-ANEPPS (Life Technologies, Carlsbad, Calif.), orANNINE-6plus (Fromherz et al., Eur. Biophys. J. 37: 509-514 (2008)).Application of an electric field to the pad results in increasedfluorescence intensity at the pad. Using voltage sensitive dyes, thefluorescence intensity of nucleic acid features clusters can be directlymodulated by applying an electric field.

Example IV Fluorescence Intensity Modulation with Quenchers Tethered toSequencing Primers

This example shows that applying an electric field at an electricallyresponsive pad that contains a probe having a quencher moiety tetheredto a fluorescent moiety can quench fluorescent signal.

The strength of quenching is dependent, at least in part, on theintermolecular distance between the quencher and the fluorophore to bequenched. There are multiple approaches to electrically modulate theinter-molecule distance between a quencher and a fluorophore that arebound to the same DNA strand. An example is shown in FIG. 7. Here aquencher is tethered to the 5′ end of a sequencing primer using a linkerarm. The 3′ end of the primer is hybridized to a target sequence and afluorophore is attached at the 3′ terminus, for example, as a result ofincorporation of a fluorescently labeled nucleotide in an SBS reaction.A quencher moiety that is appropriately charged at working pH, can beelectrically repelled or attracted towards the electrodes by a field ofopposite polarity. This results in local increase or decrease,respectively, for the quencher in the vicinity of the fluorophore. Toachieve super resolution imaging, a switch can be used to alternatelyapply electric field at two pads that are in the detection zone of asingle pixel. Fluorophores at the pads will be alternately quenchedallowing the respective target analytes to be distinguished by a singlepixel having a detection zone that includes the two pads.

Throughout this application various publications, patents and patentapplications have been referenced. The disclosures of these publicationsin their entireties are hereby incorporated by reference in thisapplication in order to more fully describe the state of the art towhich this invention pertains.

The term “comprising” is intended herein to be open-ended, including notonly the recited elements, but further encompassing any additionalelements.

Although the invention has been described with reference to the examplesprovided above, it should be understood that various modifications canbe made without departing from the invention. Accordingly, the inventionis limited only by the claims.

What is claimed is:
 1. A detection apparatus comprising: an array ofelectrically responsive pads on a substrate surface; an array ofdetection pixels, wherein each individual detection pixel in the arrayhas a detection zone on the surface, each detection pixel configured tosimultaneously observe a subset of at least two of the electricallyresponsive pads; and an activation circuit to apply electric field at afirst pad in the subset and a second pad in the subset, wherein theactivation circuit is configured to apply different electric field atthe first pad compared to the second pad, and wherein the activationcircuit comprises a switch to selectively alter the electric field atthe first pad compared to the second pad, wherein the pads comprisetarget analytes to be detected.
 2. The apparatus of claim 1, whereineach detection zone is square and each pad occurs in a corner of four ofthe detection zones.
 3. The apparatus of claim 1, wherein the targetanalytes comprise at least two nucleic acid clusters that are includedin the detection zone for a single detection pixel, each of the clusterscomprises a different nucleotide sequence from the other cluster.
 4. Theapparatus of claim 3, wherein each pad comprises a plurality of nucleicacid clusters and each cluster is included in the detection zone for asingle detection pixel.
 5. The apparatus of claim 3, wherein each padcomprises a single nucleic acid cluster and the cluster is included inthe detection zones for at least two of the detection pixels.
 6. Theapparatus of claim 3, wherein the target analytes comprise fluorescentmoieties and the detection pixels are configured to detect emission fromthe fluorescent moieties.
 7. The apparatus of claim 6, wherein theactivation circuit applies a different electric field at the first padcompared to the second pad, wherein the first pad comprises afluorescence quencher at a higher concentration than at the second pad.8. The apparatus of claim 6, wherein the activation circuit applies adifferent electric field at the first pad compared to the second pad,wherein the first pad comprises a fluorescent probe at a higherconcentration than at the second pad.
 9. The apparatus of claim 6,wherein the pads further comprise electrochemical luminescence labels.10. The apparatus of claim 9, wherein the activation circuit applies adifferent electric field at the first pad compared to the second pad,thereby producing more photons at the first pad than at the second pad.11. The apparatus of claim 1, wherein the activation circuit isconfigured to apply an electric field at the first pad while no field isapplied at the second pad, and the switch is configured to turn off theelectric field at the first pad while applying an electric field at thesecond pad.
 12. The apparatus of claim 1, wherein the activation circuitis configured to apply a positive electric field at the first pad whileapplying negative electric field at the second pad, and the switch isconfigured to apply negative electric field at the first pad whileapplying positive electric field at the second pad.
 13. A method ofdetecting analytes, comprising: providing the detection apparatus ofclaim 1, wherein the two pads comprise different target analytes,respectively; acquiring signals from each of the detection pixels whileselectively applying an electric field at a first of the two pads topreferentially produce signal from a first of the different targetanalytes compared to a second of the target analytes, therebypreferentially acquiring signals from the first of the target analytescompared to the second of the target analytes; and acquiring signalsfrom each of the detection pixels while selectively applying an electricfield at the second of the two pads to preferentially produce signalfrom the second of the different target analytes compared to the firstof the target analytes, thereby preferentially acquiring signals fromthe second of the target analytes compared to the first of the targetanalytes.
 14. The apparatus of claim 1, wherein the array of pads hasthe same pitch as the pitch for the array of pixels.
 15. A methodcomprising: providing a detection apparatus of claim 1, simultaneouslyobserving a first pad and a second pad using a detection pixel; applyinga first electrical field at the first pad; and applying a secondelectrical field at the second pad, the first electric field differentthan the second electric field, the first pad and the second padcomprising target analytes to be detected.